Research in Progress Currently, there are three main ongoing projects in the lab: The first project is focused on elucidating mechanistic details of the interaction between type II topoisomerases and DNA. One aspect of this interaction concerns the ability of type II topoisomerases to relax the topology of DNA to below equilibrium values. In vivo these topoisomerases are responsible for unlinking replicated chromosomes prior to cell division. Since even a single link between sister chromosomes can prevent division and induce cell death, it is important that these enzymes preferentially unlink rather than link DNA molecules. In vitro it was shown that this is the case, but the mechanism remains a mystery. Previously we have shown that a mechanism based on a sharp bend imposed on the DNA by the topoisomerase cannot explain the extent of non-equilibrium simplification, and cannot explain the differences in non-equilibrium simplification among different type II topoisomerases (bacterial, human, yeast). We have recently completed testing two alternative models of topology simplification. The models postulate either a kinetic proofreading mechanism in which the topoisomerase catalyzes strand passage only after repeatedly encountering a DNA segment, or a mechanism in which the topoisomerase specifically recognizes DNA in a hooked juxtaposition geometry. Our recent measurements indicate that type II topoisomerases can catalyze DNA strand-transfer with each collision of two DNA segments, thereby ruling out the kinetic proof reading model. Furthermore, preliminary evidence suggests that DNA unlinking rates are not highly correlated with the degree of hookedness of the two strands. Further tests, currently underway, will allow us to unambiguously determine the validity of the hooked juxtaposition model in describing the activity of type II topoisomerases. More recently we proposed an alternative model of non-equilibrium topology simplification in which the preferential relaxation of supercoiled DNA arises from preferential binding of the type II topoisomerases to more supercoiled substrates, i.e., a topology-dependent binding. To test this model we first developed a novel assay to measure DNA topology-dependent protein binding. We have validated this assay with a number of DNA binding proteins. Using this assay, we have identified a significant influence of DNA topology on the binding of type II topoisomerases to DNA, consistent with the premise of our model. Thermodynamic modeling of the relaxation process suggests that the topology-dependent binding contributes to the non-equilibrium relaxation process, but we have not as of yet achieved quantitative agreement with experimental measurements of non-equilibrium relaxation. Further experimental and theoretical work is ongoing to resolve the remaining quantitative discrepancies. Nonetheless, this work has led to the development of a novel and very general new protein-DNA binding assay and has uncovered a novel mode of action of type II topoisomerases. The second project is focused the mechanisms underlying multi-enzyme complex activity. RecQ helicases and topoisomerase III have been shown to functionally and physically interact in organisms ranging from bacteria to humans. Disruption of this interaction leads to severe chromosome instability, however the specific activity of the enzyme complex is unclear. Analysis of the complex is complicated by the fact that both the helicase and the topoisomerase individually modify DNA. The ability of single-molecule techniques to measure the activity of a single enzyme or enzyme complex in real time is well suited to the study of such complicated processes in which multiple activities may occur over multiple time scales. In collaboration with Mihaly Kovacs at Etovos University, Hungry, we are using single-molecule measurements of DNA unwinding to elucidate the kinetics and step size of RecQ helicase alone and in the presence of Topo III. These experiments will pave the way for experiments in which the activity and the association state of single enzymes and complexes will be assayed simultaneously using a combination of single molecule manipulation and single molecule visualization techniques. The third related project, in collaboration with Yves Pommier in NCI, is focused on the mechanisms of supercoil relaxation by human type IB topoisomerases, and in the effects of chemotherapy agents that inhibit type IB topoisomerases. Type IB topoisomerases are essential enzymes that relax over wound (positively supercoiled) DNA generated ahead of the replication machinery during DNA synthesis. Type IB topoisomerases are also important chemotherapy targets. Potent chemotherapy agents that specifically inhibit type IB topoisomerases are currently in clinical use and additional agents are in development. We are using single-molecule magnetic-tweezers based assays to measure the activity of individual type IB topoisomerases and the effects of chemotherapy agents on the activity. These experiments provide molecular level details of the supercoil relaxation process and how it is influenced by the degree of DNA supercoiling, the tension on the DNA, and the presence of specific chemotherapy agents. These measurements provide an unprecedented level of detail concerning how these important enzymes work and are inhibited by chemotherapy agents. We recently demonstrated that the human nuclear Topoisomerase IB is remarkably insensitive to the effects of twist or torque on the DNA. This observation, combined with the first direct measurement of the cleavage kinetics at the single-molecule level, allowed us to formulate a comprehensive model for the complete relaxation and religation process catalyzed by type IB topoisomerases. This model reveals a hitherto unobserved intermediate state in the relaxation cycle, and provides a mechanistic framework for the action of inhibitors. More recently, we have employed the single-molecule supercoil relaxation assay to characterize the molecular consequences of a unique mitochondrial topoisomerase I specific inhibitor, Lamellarin-D, a natural product derived from a mollusk. Binding of the inhibitor to the topoisomerase results in DNA topology-dependent changes in the rate of supercoil relaxation and inhibition of religation. We have recently extended these measurements to compare the mechanistic consequences of nuclear topoisomerase I inhibition by four different inhibitors corresponding to three classes of compounds. The single-molecule results reveal significant topology-dependent changes in the effects of the inhibitors on the topoisomerase. These measurements also provide a complete kinetic characterization of inhibitor binding to topoisomerases engaged in relaxation of defined topological states, which have implications for the mechanism and efficacy of inhibition in vivo. These projects have been enabled by the development of a unique magnetic tweezers instrument that affords high spatial and temporal resolution measurements of DNA topology combined with real-time computer control and position stabilization. The ongoing development and improvement of this magnetic tweezers instrument represents a sustained research endeavor. Future research goals: Our immediate goal is the completion of the ongoing projects in the lab. Longer term goals include the development of a new optical trap and magnetic tweezers instrument combined with single-molecule fluorescence detection.